This invention relates to an isolated bacteriophage and a process for preparation of said bacteriophage useful as a tool for studying biological, biochemical, physiological and genetic properties of actinomycetes and other organisms. The bacteriophage obtained by the present invention is particularly useful for the characterization of antibiotic biosynthetic pathways and similar primary and secondary metabolic pathways. The bacteriophage described in this process may also be used for the study of several other genetic and physiological pathways. It may also be used to generate mutations in the metabolic pathways of bacteria, and after some specific alteration such as after cloning into suitable mobilising vectors, may also be used to study the metabolic pathways of other microorganisms and plants. Mutations can be generated essentially at any locus of the bacteria. The results of the above mentioned alterations or mutations may lead to the production of new metabolic products or to the expression of new physiologically active compounds or to the expression of novel characteristics either in the same host or in heterologous hosts.
As used in this application, bacteriophage or phage refers to a virus or viruses which infect bacteria. After infection the virus can produce new progeny particles or can remain dormant within the bacterial genome. Lysogen is a bacteria which carries a bacteriophage without being harmed. Plaque is a turbid/clear spot produced due to lysis of the cells infected by a bacteriophage. Prophage is a bacteriophage that is maintained in the lysogenic state in a bacterial cell. Confluent lawn is a uniform growth of organism on an agar plate. Phagemid is a plasmid vector carrying some part of the phage DNA. The term restriction enzyme refers to an endonuclease that cuts DNA sites defined by its recognition sequence. Auxotroph is a bacterial strain defective for the synthesis of one or more sugar or amino acids. Restriction barrier is intended to mean a host defense system which protects the bacteria from invaders, by cleaving the invaders"" DNA. Cloning is understood to mean the procedure for the generation of recombinant DNA. Genetic tool is a system where DNA is used as a tool. Mutation is an alteration in the sequence of bases in the DNA of an organism. This alteration may be caused by insertion, deletion or modification of DNA bases. Transposon refers to a genetic element that carries the information that allows it to integrate at various sites in the host genome. Genome is a term used to describe the complete genetic complement of a virus, or cell or bacteria or any living organism.
The process of the present invention involves the isolation from soil samples and purification of a novel bacteriophage from a strain of Saccharomonospora called PA136. The strain of Saccharomonospora described in the present invention is characterized by the presence of single lateral white coloured spore, meso-diaminopimelic acid; arabinose and galactose as total sugar but no mycolic acid and major components of fatty acids-16 carbon iso- and antiso fatty acids. Other properties of this strain are: it grows on hypoxanthine, hypoxanthine+0.3% glucose, tributyrine, xylitol, thallous acetate, thallous acetate+0.1% glucose, propanol, butanol-1, 3 diol, D-fucose, salicin, arabinose, L-asparagine, phenylalanine, L-serine, and sodium benzoate. The strain grows in the temperature range of 20xc2x0 C. to 55xc2x0 C. on an agar plate with pH range 5.5 to 9.0. It is catalase positive and produces extracellular enzymes: lipase (C14), leucine arylamidase, valine arylamidase, cystein arylamidase, trypsin, chemotrypsin, acid phosphatase, naphthol As-B1-phosphophydrolase, xcex1-glucosidase and xcex2-glucosidase. The chemical composition of the total cell is as follows: meso-di-aminopimelic acid, arabinose, galactose, phosphatidyl glycerol, di-phosphatidyl glycerol, phosphatidyl ethanolamine, hydroxyphastidyl ethanolamine, phosphatidyl inositol glycolipids are present. Sugar containing unidentified groups of phospholpids is present. Total fatty acids are: major components of 16 carbon iso- and antiso-fatty acids of branched and straight chain carbon compounds. It does not utilise sucrose. It mostly produces a diffusable pigment that is either green, orange or yellow. Sometimes the strain Saccharomonospora PA136 does not produce any pigment at all. At times the pigment is non-diffusable. However, the pigments produced by the strain Saccharomonospora PA136 are water-soluble. The pigments are insoluble in ether, ethanol, methanol, butanol, isopropanol, benzene, ethyl acetate, chloroform and acetone. It is partially soluble in phenol. The green pigment produced by the strain is a characteristic of the genus Saccharomonospora. The strain Saccharomonospora PA136 undergoes autolysis after 5-6 days of incubation on a culture plate which when subjected to detailed analysis results in the isolation of a bacteriophage named as PIS136. This temperate bacteriophage has a wide host range amongst Gram positive (Gram+) bacteria and generates lysogens at the rate of 2 to 3 percent of the total cells infected. The phage PIS136 has a DNA genome of about 90 kb where the GC (Guanidine and Cytosine) content is 69 to 71 mol percent. The genome of this phage is partially methylated and lacks recognition sites for many restriction enzymes. The phage genome or bacteriophage that has the property of generating random mutations by transposition also shows the phenomenon of gene inversion. The phenomenon of gene inversion can be used to control the host range of the phage as well as for heterologous and conditional expression of genes. The phage has been deposited as a lysogen of the strain Saccharomonospora PA136 at the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh and carries an accession number MTCC A0001, where xe2x80x98Axe2x80x99 stands for Actinomycetes and also bears the depository number DSM 12317 at DSMZ-DEUTSCHE SAMMELUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH where it was deposited on Jul. 16, 1998. Because of the unique properties this phage has immense potential as a genetic tool and can be used variously as a transposon for the generation of mutants, as an intergeneric cloning vector, for the study of metabolic pathways, as a reporter phage, for conditional gene expression, or even for the activation of silent genes, etc.
Bacteriophages or phages, the viruses of bacteria, are the simplest of all living organisms. They have been natural objects of study in attempts to understand life at the molecular level. Phages have evolved a number of regulatory schemes to ensure efficient production of progeny particles during development. However, in general, in about 1% of the cells that are infected by the temperate phages, lysogeny is established, that is when the phage genome integrates with the host. The lysogens are immune to super-infection.
Numerous phages have been isolated from industrially and medically important bacteria such as Streptomyces species, Corynebacterium species, Lactococcus lactis, Mycobacteria, Escherichia coli, Salmonella and Staphylococcus species etc.
About 70% of the known and naturally occurring antibiotics are produced by members of the genus Streptomyces. In most cloning procedures for Streptomyces species, plasmid based vectors capable of replicating autonomously have been used (Rao, R. N., Richardson, M. A. and Kuhtoss, S. 1987 in Methods in Enzymology. 153: 166-198; Hopwood, D. A., Bibb, M. J., Chater, K. F. and Kieser, T. 1987 in Methods in Enzymology. 153: 116-165).
At the same time the relatively broad host range temperate bacteriophage ØC31 (which infects Streptomyces species and the related genus Streptoverticillium species only) was developed as a versatile cloning vector (Chater, K. F. 1986, In The Bacteria, Vol. IX. Queener, S. W. and Day, L. E. (ed) London: Academic Press pp 119-158; Kobler, L., Schwertfirm, G., Schmieger, H., Bolotin, A. and Sladkova, I. 1991 FEMS Microbiology Letters, 8: 347-354, Bruton, C. J., Guthrie, E. P. and Charter, K. F. 1991. Bio/Technology, 9: 652-656). In 1991, Kuhtoss, S., Richardson, M. and Rao, R. N. (Gene, 97: 143-146) developed a shuttle vector which utilizes the site specific recombination system of the phage ØC31. The vectors allow the cloned DNA to be stably inserted into a host cell. McHenny, M. A. and Baltz, R. H. (Journal of Bacteriology, 1988 170: 2276-2282) claimed to have developed a transduction system for several species of Streptomyces and related genera using the phage FP43 but this system failed on a very important strain of Streptomyces hygroscopicus 10-22. Thus Zhou, X., Deng, Z., Hopwood, D. A. and T. Kieser, (1994 Journal of Bacteriology 176: 2096-2099) used a new phage ØHAU3 which has a relatively broad host range within Streptomyces and developed a phagemid to study the molecular biology of the Streptomyces hygroscopicus strain 10-22.
Phages of another industrially important organism, Lactococcus lactis have also been exploited for vector development. (Kok, J., Van der Vossen, J. M. B. M. and G. Venema. 1984 Applied Environmental Microbiology, 48: 726-731; Vander Vossen, J. M. B. M., Van der Lelie, D. and Venema, G. 1987 Applied Environmental Microbiology, 53: 2452-2457). Attempts have also been made to develop the L5 phage of mycobacteria into a suitable vector in order to simplify the study of the molecular biology of mycobacteria (Lee, M. H., Pascopella, L., Jacobs, W. R. and G. F. Hatfull. 1991 Proc. Natl. Acad. Sci. USA, 88: 3111-3115.; Donnelly-Wu, M. K., Jacobs, Jr. W. R. and G. F. Hatfull. 1993 Molecular Microbiology, 7: 407-417). In spite of all these studies, the application of molecular techniques has not yet led to dramatic results in industrial strain development programmes or in the development of hybrid Or new antibiotics. However, none of the phages of Gram positive (Gram+) bacteria that have been reported in the literature have the property of being a transposon.
A transposon has the property that it can insert itself at random sites within the host genome. By virtue of their insertion properties, transposons have been used to improve the production of secondary metabolites and to construct strains that will produce hybrid or new secondary metabolites. Transposons have also been used for gene disruption and to clone global regulatory genes, pathways of secondary metabolites and strong or regulatory promoters. They might also be used to block competing or unnecessary pathways in order to achieve more efficient production of secondary metabolites (Hahn, D. R., Solenberg, P. J., McHenny, M. A. and R. H. Baltz. 1991, Journal of Industrial Microbiology, 7: 229-234).
The present invention is thus based upon the need to develop a system with which one may be able to study the molecular biology of the class of Gram+ bacteria which contains a large number of organisms, such as species of Amycolatopsis, Corynebacterium, Mycobacteria, Nocardia, Micromonospora, Rhodococcus, Streptomyces etc. which are of both industrial and medical importance. At present there is no known multipurpose transposon or phage mediated vector or generalised transduction system available with which the genetics and molecular biology of these organisms can be studied without any problem. Further, most of these organisms are industrially very important because they produce antibiotics, enzymes, small peptides which are bio-active, glutamic acid and many other amino acids as secondary metabolic products. The regulation of these pathways is extremely complicated as several intermediates and precursor compounds are required for biosynthesis. In general, the secondary metabolic pathways within an organism are interrelated in that either they use same precursor molecule or one uses an intermediate compound of other as precursor. This may result in the poor production of the desired end product of one of the pathways compared to its related pathway. As the production level is very low with natural isolates, the goal of obtaining a high producer strain presents a major challenge to industry. Furthermore, most of the natural isolates also produce more than one secondary metabolite and the presence of another compound is often undesirable. Thus a bacteriophage having a property which can be used to stop the production of undesirable compounds by mutation will stop the diversion of a precursor compound from one pathway to another, which is often a limiting factor, will be very useful to industry. The property of gene inversion in the bacteriophage/phage described in the present invention may be used to overcome the problems that arise when an intermediate product of one pathway is a precursor molecule in other pathway. The process herein will be that that the inverting fragment will be cloned in one of the biosynthetic pathways in such a way that when the inverting fragment is in one orientation the pathway will function normally, however, when it is in the other orientation the biosynthesis will be stopped at the point at which the inverting fragment is inserted. However, other part of the pathway that is functioning normally will allow the accumulation of intermediate products that may be used by some other biosynthetic pathway. Since the process of inversion can be externally controlled, it may be possible to modulate the biosynthetic pathways as per need. Thus the bacteriophage described in the present invention will bring a revolution, particularly to those industries that are involved in the commercialization of useful secondary metabolites produced by the microorganisms. This phage also infects mycobacteria and mutants of mycobacteria can be obtained. These mutants may be used as live vaccines. Thus this invention establishes the possibility to have a transposon-phage which can be exploited as a generalized transduction system for several genera of Gram+ bacteria.
In the preparation of the bacteriophage, the choice of the growth medium is very important. Several known media such as (1) Luria Bertani (LB) having the composition per 1000 ml: 10 gm Tryptone, 5 gm Yeast extract, 10 gm Sodium Chloride; (2) Yeast extract: malt extract per 1000 ml: 2 gm Yeast extract, 4 gm Malt extract, 10 gm Glucose; (3) Tryptic Soya broth per 1000 ml: 17 gm Pancreatic Digest of casein, 3 gm Papaic digest of Soya meal, 5 gm Sodium Chloride, 2.5 gm Dibasic Potassium Phosphate, 2.5 gm Dextrose and (4) Bennett""s medium containing per 1000 Nm: 1 gm yeast extract, 1 gm Beef Extract, 2 gm Pancreatic digest of casein, 10 gm Dextrose, were tested and found that none of the media favoured the release of large amount of phage particles.
The components of the known media listed above are not easily assimilable and do not appreciably favour the lytic cycle of the phage. Further, all the above media lack trace elements that are very important for the growth of live organisms which ultimately may affect the lytic cycle of the bacteriophage. Accordingly, one embodiment of this invention relates to a growth medium, useful, inter alia, for fungi and bacteria, particularly, strain of Saccharomonospora called PA 136 which is a lysogen of the phage PIS 136.
The main object of the invention therefore, is to prepare a bacteriophage that may be useful as a genetic tool for studying the biological, biochemical, physiological and genetic properties of a large number of actinomycetes genera and other Gram+ bacteria. The process involves steps such as isolation and purification of a novel temperate bacteriophage (or phage), from a species of Saccharomonospora called PA136 and where either the phage or its bacteriophage can be used as a genetic tool to study the molecular biology and other properties of a large number of Gram+ bacteria. The species Saccharomonospora PA136 is a lysogen and upon lysis yields the wide host range mutagenic bacteriophage PIS136.
Another object of the present invention is to use the bacteriophage described herein, or the phage as a transposon and then in the generation of random mutations in a wide range of bacteria or any other suitable host.
Yet another object of the present invention is to use the invertible fragment of the phage cloned into a suitable vector to control/regulate the secondary metabolic pathways either of the host strain or of a heterologous host. The phage PIS136 can overcome the restriction barriers of many of the actinomycetes strains and establish itself as a lysogen by integrating into the host genomes at random sites.
It is yet another objective of this invention to prepare a universal growth medium which supports the growth of a large number of organisms belonging to various groups such as fungi, Gramxe2x88x92 and Gram+ bacteria, particularly, strain of Saccharomonospora PA 136.
It is yet another objective of this invention to develop a medium which can shorten the time required for growth, which can produce large amount of exponentially growing cells and also which can withstand relatively high temperature of growth without having an adverse effect on the growth pattern.
It is another object of the invention to provide a growth medium that possesses essential trace elements necessary for growth of live organisms.
It is yet another object of the invention to provide a medium that induces the lytic growth of bacteriophage, particularly the phage PIS 136.
Accordingly, the present invention provides a isolated bacteriophage useful as a tool for studying biological, biochemical, physiological and genetic properties of actinomycetes and other organisms which comprises incubating a novel strain of Saccharomonospora having characteristics as herein described.
The present invention also provides a process for the isolation of a bacteriophage as a tool for studying biological, biochemical, physiological and genetic properties of actinomycetes and other organisms which comprises incubating a novel strain of Saccharomonospora having characteristics as herein described in a nutrient medium such as herein described until the autolysis stage is reached, isolating and purifying from the said medium a bacteriophage generated in the lysed culture by conventional methods, then breaking-up the proteinaceous envelop of the said isolated bacteriophage by known methods followed by recovering the bacteriophage by conventional precipitation techniques.
The present invention also provides a novel growth medium and a process for the preparation thereof useful for fungi and bacteria, particularly, strain of Saccharomonospora PA 136.
In an embodiment of the present process the subject matter a microorganism is a novel isolate of the genus Saccharomonospora and has taxonomic characteristics typical of the genus.
In another embodiment of the present process the novel isolate may be grown in any medium comprising varying quantities of beef extract, yeast extract, tryptone, tryptose, peptone, proteose peptone, malt extract, glucose, calcium chloride, magnesium chloride, ferric ammonium citrate, cobalt chloride at pH 7.0 to 7.5. However, the preferred medium comprises beef extract, tryptose, proteose peptone, yeast extract, glucose, calcium chloride, magnesium chloride, ferric ammonium citrate, cobalt chloride at pH 7.2, on quantities as particularly as described herein.
In yet another embodiment the isolate upon incubation for 3-6 days helps the release of the phage PIS136 in to the medium. However, the best result are obtained from fifth day onwards.
In another embodiment the isolated bacteriophage has about 90 kilobase pairs of lond double stranded DNA genome with guanidine and cytosine content of 69-71 percent.
In yet another embodiment the phage may be purified by using a modified procedure of Yamamoto et al., 1970 and Smorawinska et al., 1988 (Yamamoto, K. R.; Alberts, B. M., Benzinger, R., Lawhorne, L. and Treiber, G., 1970 Virology, 40: 734-744; Smorawinska, M., Denis, F., Dery, C. V., Magny, P. and Brzenski, R. 1988. Journal of General Microbiology, 134: 1773-1778).
In another embodiment, the invention also relates to a cloning vector which comprises a DNA molecule of the phage, and may comprise a plasmid or a bacteriophage.
In yet another embodiment the phage so obtained may be used to generate mutations in the metabolic pathways of bacteria and after some specific alteration such as after cloning in to suitable mobilising vectors, may also be used to study the metabolic pathways of other micro organisms and plants. The results of the above mentioned alterations or mutations may lead to the production of new metabolic products or to the expression of new physiolocial active compounds or may change the pathogenic property of an organism but maintain the immunogenic property which would enable the organism to be used as a live vaccine strain or to the expression of novel characteristics either in the same host or in heterologous hosts.
The novel growth medium of the invention is prepared by mixing the appropriate quantities of the following chemicals and complex media components such as: Beef extract or Lab Lamco, Yeast extract, Tryptose, Proteose peptone, Soluble starch, Dextrose, and traces of Cobalt chloride or Cobalt nitrate and Ferric ammonium citrate. The composition when established and dissolved in double glass-distilled water, at room temperature, has a pH from about 6.0 to 6.6. This medium when used as it is, or after adjusting the pH between 7.0 and 7.2 by using IN Sodium hydroxide acts as a universal medium for the growth of large number of actinomycetes genera, other Gram+ bacteria, some Gramxe2x88x92 bacteria and for several groups of fungi. In particular, it was useful as an excellent medium for the growth of strain of Saccharomonospora PA 136.
Thus, the present invention provides for a process for the preparation of a universal growth medium useful for fungi and bacteria which comprises mixing of A, B and C, i.e. macronutrients, micronutrients and trace elements in the quantities mentioned as below:
A. macronutrients in the form of assimilable carbon sources such as:
1. Lab Lamco or Beef extract 8-12 gm
2. Starch 1-2 gm
3. Dextrose 10 to 12 gm and
also in the form of assimilable nitrogen sources such as:
1. Protease peptone 1-3 gm
2. Tryptose 1-3 gm
B. micronutrients selected from yeast extract 1-3 gm, a and
C. trace elements selected from:
1. Cobalt chloride or nitrate 5-10 mg
2. Ferric ammonium citrate 5-10 mg
3. Magnesium sulphate or chloride 0-10 mM
4. Calcium chloride or nitrate 0-10 mM;
and thereafter making the volume to 1000 ml by the addition of double-distilled water.